One of the goals of molecular biology is to understand the biological information contained in DNA and RNA sequences. The use of synthetic, sequence defined DNA and RNA has played a key role in understanding the genetic code and various regulatory signals such as the operator, promoter, ribosomal binding sites, enhancers, and transposable elements. The synthetic approach not only provides a final proof of the roles of various DNA and RNA sequences but also offers an opportunity for further improvement in function for practical application. The application of synthetic genes, linkers, primers, and probes from both DNA and RNA has become a powerful tool in the cloning, sequencing, and isolation of genomic DNA.
The synthetic methodology for the synthesis of short oligoribonucleotides by the phosphodiester approach was developed in the 1960s by Khorana, H. G., Pure Appl. Chem. 17:349-381 (1968). Organic chemical syntheses of larger molecules of oligoribonucleotides have been attempted by using the phophodiester, phosphotriester, or phosphite triester methods. However, the discovery of RNA ligase has extended the possibilities for synthesizing RNA molecules such as tRNA. Before RNA can be synthesized, however, the starting monomers must be provided. In synthesizing sequence-defined RNA oligomers, the purity and correct structure of the monomer building blocks is critical.
Currently, reports are available on the synthesis of N-protected-2'-O-methyl-3'-O-chlorophenyl phosphotriester ribonucleosides (Inoue, H. et al., FEBS Letters, 215:327-330 (1987) "Inoue I") and N-protected-2'-O-methyl-3'-cyanoethyl phosphoramidite ribonucleosides (Sproat, B. S. et al., Nucl. Acids Res. 18:41-49 (1990)). The methyl iodide/silver oxide method developed by Inoue et al. (1987) on the seven membered bis-sililoxy protected (Markiewicz, W. T., J. Chem. Res. (S): 24-25 (1979)) uridine and N.sup.6 -cytidine generates the undesired 3'-O-methyl-isomers to the extent of 6-8%.
It is believed that methyl iodide in the Inoue method causes the partial ring opening, and subsequently leads to 3'-O-methyl-isomer formation. Similarly, the CH.sub.2 N.sub.2 reaction under very mild reaction conditions still leads to formation of some 3'-O-methyl isomer (4-5%). This reaction is described in Inoue et al. (1987), as well as in Ekborg, G. et al., J. Carbohyd. Nucleosides and Nucleotides, 7:57-61 (1982) and Heikkila, J. et al., J. Acta. Chem. Scand., B36:715-717 (1982).
It is therefore important to establish stringent purification techniques and a certain homogeneity of the phosphoramidites because of their use in RNA syntheses. In Sproat, B. S. et al., Nucl. Acids Res. 18:41-49 (1990)), discussing the synthesis of N-protected-2'-O-methyl-3'-cyanoethyl phosphoramidite ribonucleosides, the authors report only a single peak in .sup.31 P-NMR of N.sup.2 -(4-tertbutyl) benzamido-2'-O-methyl-3'-cyanoethyl phosphoramidite of guanosine.
Since these products are used directly in the synthesis of defined sequence RNA, the purity as well as absolute structure assignment is very critical for any biological application. Besides the concern for purity of these biochemicals, it is also advantageous to develop products which have the most commonly used protecting groups on the pyrimidine or purine ring system of these monomers. This is important for the convenience of the synthesizer and dramatically improves the quality of the 2'-O-methyl-RNA oligomers produced from these monomers. Thus, the N.sup.2 isobutyryl group on guanosine and N.sup.6 benzoyl on adenosine represent the most versatile protecting groups for the aforementioned purposes.